A scallop spermatogonia surface marker protein and its application in genetic breeding

Abstract

The present invention provides a scallop spermatogonia surface marker protein and its application in genetic breeding. The amino acid sequence of the scallop spermatogonia surface marker protein is SEQ ID NO: 1. The present invention also provides a use of the scallop spermatogonia surface marker protein, which is used as a specific marker in marking scallop spermatogonia. In another aspect, the present invention also provides a method for detecting scallop spermatogonia, wherein the method uses an immunohistochemical method to detect whether the above-mentioned scallop spermatogonia surface marker protein is present. The present invention screened and obtained a scallop spermatogonia surface marker protein, which is used as a scallop spermatogonia-specific marker, thereby laying the foundation for the separation and in vitro culture of scallop spermatogonia.

Description

Technical Field

[0001] The invention belongs to the technical field of marker proteins and genetic breeding, and particularly relates to a scallop spermatogonia surface marker protein and application thereof in genetic breeding. Background Art

[0002] Spermatogonia are crucial for spermatogenesis and male reproduction. In vertebrates, there are three main types of spermatogonia: undifferentiated, differentiating, and differentiated spermatogonia. Spermatogonial stem cells are undifferentiated spermatogonia that have the ability to self-renew and maintain spermatogenesis. Spermatogonial stem cells account for a very low proportion in the adult testis (0.3% in mice, 4% in monkeys, and 22% in humans), and the primary task of their research is isolation and enrichment.

[0003] Current research typically uses Percoll gradient density centrifugation, differential adhesion, or fluorescence-activated / magnetic-activated cell sorting (FACS) to enrich spermatogonial stem cells. Fluorescence-activated / magnetic-activated cell sorting (FACS) has been shown to yield spermatogonial stem cells with higher purity than other methods. However, the use of these two methods is limited by the availability of spermatogonial stem cell-specific surface markers. To date, spermatogonial stem cell surface markers have been extensively studied in many vertebrate species, but many spermatogonial stem cell markers are not conserved across organisms. Therefore, it is imperative to develop and screen spermatogonial stem cell surface markers in the species of interest.

[0004] Molluscs are the second largest group of invertebrates, encompassing over 100,000 species. Most mollusks have lengthy reproductive cycles, making them highly vulnerable to natural disasters, infection by pathogenic microorganisms, and other environmental threats. Isolating germline stem cells for cryopreservation and in vitro differentiation holds promise for shortening the reproductive cycle of shellfish, preserving germplasm resources, and promoting the sustainable development of shellfish aquaculture. Compared to primordial germ cells, which originate from earlier development, spermatogonial stem cells originate from the highly cellular adult gonad and are therefore easier to isolate.

[0005] However, our understanding of molluscan germline stem cells remains limited, and techniques for manipulating germline cells (such as isolation, enrichment, culture, and gene editing) are still in their infancy. There are no surface markers for identifying spermatogonial stem cells or spermatogonia. Therefore, developing specific surface markers for scallop spermatogonia will facilitate the characterization and manipulation of molluscan spermatogonia and provide useful screening markers for shellfish genetic breeding. Summary of the Invention

[0006] The invention aims to provide a scallop spermatogonia surface marker protein and its application in genetic breeding, namely, a marker gene Fgfr discovered through screening and its application.

[0007] The present invention first provides a scallop spermatogonia surface marker protein, comprising:

[0008] 1) a protein having an amino acid sequence of SEQ ID NO: 1;

[0009] 2) Proteins derived from 1) by replacing, deleting, or adding one or more amino groups in 1).

[0010] A nucleotide sequence of the gene encoding the scallop spermatogonia surface marker protein is SEQ ID NO: 2.

[0011] The present invention also provides a recombinant expression vector for recombinantly expressing the scallop spermatogonia surface marker protein;

[0012] Furthermore, a nucleotide fragment with the sequence of SEQ ID NO: 4 is inserted into the recombinant expression vector.

[0013] The present invention also provides a recombinant expression engineering strain, which is transformed into the above-mentioned recombinant expression vector.

[0014] In another aspect, the present invention provides a use of the scallop spermatogonia surface marker protein, which is used as a specific marker in marking scallop spermatogonia.

[0015] The present invention also provides a product for marking scallop spermatogonia, which contains antibodies for detecting the surface marker protein.

[0016] Another aspect of the present invention provides a method for detecting scallop spermatogonia, wherein the method uses an immunohistochemical method to detect whether the above-mentioned scallop spermatogonia surface marker protein is present.

[0017] The present invention screened and obtained a scallop spermatogonia surface marker protein, which is used as a scallop spermatogonia-specific marker, thereby laying a foundation for the separation and in vitro culture of scallop spermatogonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the single-cell expression pattern and cell localization map of the scallop Fgfr gene. The results show that the Fgfr gene is specifically expressed in spermatogonia.

[0019] Figure 2The figure shows the expression of recombinant protein of the extramembrane region of scallop FGFR protein under different conditions, where lane 1: total bacterial protein before IPTG induction; lanes 2-3: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 4℃ for 16h; lanes 4-5: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 8℃ for 16h; lanes 6-7: supernatant of bacterial lysate after induction with 0.2mM and 0.5mM IPTG at 12℃ for 16h.

[0020] Figure 3 This is the SDS-PAGE gel electrophoresis diagram of the purification of the recombinant protein of the extramembrane region of scallop FGFR protein, where lane 1: total bacterial protein before IPTG induction; lane 2: supernatant of bacterial lysate after induction with 0.5 mM IPTG at 12°C for 16 hours; lane 3: flow-through of Ni-NTAResin affinity purification; lanes 4-12: wash solution; lane 13: eluate.

[0021] Figure 4 This is the standard curve for the BCA determination of the recombinant protein concentration of the scallop FGFR protein extramembrane region, with a protein concentration of 0.55 mg / mL.

[0022] Figure 5 This is a Western Blot analysis of scallop FGFR recombinant protein antibodies, where lane 1: Anti-his rabbit monoclonal antibody is used to detect scallop FGFR recombinant protein; lane 2: Anti-FGFR rabbit polyclonal antibody is used to detect scallop FGFR recombinant protein; lane 3: Anti-FGFR rabbit polyclonal antibody is used to detect scallop testis FGFR protein.

[0023] Figure 6 This is the immunohistochemical validation diagram of the scallop FGFR recombinant protein antibody. The results show that the scallop FGFR recombinant protein rabbit polyclonal antibody specifically detects spermatogonia.

[0024] Figure 7 This is the immunofluorescence validation image of the scallop FGFR recombinant protein antibody. The results show that the scallop FGFR recombinant protein rabbit polyclonal antibody specifically binds to the spermatogonial cell surface protein. DETAILED DESCRIPTION

[0025] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with the accompanying drawings and specific examples. Unless otherwise specified, the equipment and experimental methods used in the present invention are all conventionally purchased reagents, equipment and methods of use.

[0026] Example 1: Screening of scallop spermatogonia surface marker protein genes

[0027] 1) A single-cell transcriptome library was constructed from the gonads of scallops at the early stages of sexual differentiation. Cell populations were then classified through data processing. Spermatogonia were identified through expression patterns and functional enrichment analysis of marker genes in different populations.

[0028] 2) Screen receptor genes from spermatogonial cell marker genes, obtain the spermatogonial cell surface candidate marker Fgfr, and design and synthesize sense and antisense probes.

[0029] 3) In situ hybridization was performed on mature scallop testis samples to determine the cellular localization of the Fgfr gene. Paraffin sections of the testis were mounted on slides, rehydrated with graded alcohol, and then incubated with 2μg / mL proteinase K in PBST (PBS containing 0.1% Tween 20) at 37°C for 15 minutes. After prehybridization in hybridization buffer at 60°C for 3 hours, hybridization was carried out overnight with 1μg / mL RNA probe at the same temperature. After removing the probe, the sections were incubated in blocking buffer (PBST and 0.5% blocking reagent) for 1 hour and incubated with 1:2000 anti-digoxigenin antibody (Roche, Germany) at 4°C for 16 hours. After being thoroughly washed with maleic acid buffer (0.1mol / L maleic acid, 0.15mol / LNaCl, 0.1% Tween-20, pH=7.5), the sections were incubated with NBT / BCIP solution (Roche, Germany) for color development. Finally, the sections were counterstained with 1% neutral red solution. Results are shown in Figure 1 .

[0030] The amino acid sequence of the extracellular region fragment protein of the Fgfr gene, a surface marker of scallop spermatogonia, obtained by screening is as follows:

[0031] NDLKVYEGDKARLRCVVKGHPRPWIAWTKNNQSLTPKQDPRYKINRNALVIDKVHRNDTAVFSCFAWNELGNISTTFTLTVITEEEEEPVEQSYAISKCNKKEEGAPIWKTSKENSRWIARPARAPVELKCQACGNPIPNITWYKNSQIIDSSVKEGKYQVDGYKL KMTDLTTEDNGNYTCMIQNRYGKLMWMYMLEVIDRNVQKPDVEGPVNTTAYIGENVKLHCKVKSDIQAHIQWVKHYQINGTYRKPDGTPYVHVIQQSTYNMTNPEILEIINVSRSDTGWYTCLVSNFAGRRFGSAWLTVEDVPGNESLSNMRHAQEAPRSSAK(SEQ ID NO:1);

[0032] The nucleotide sequence of its coding gene is as follows:

[0033] AATGATCTGAAAGTTTACGAAGGTGATAAAGCGCGTCTGCGTTGCGTTGTTAAAGGCCACCCGCGTCC

[0034] GTGGATTGCGTGGACCAAAAACAACCAGAGCCTGACCCCGAAACAAGATCCGCGTTACAAAATCAACCGTA

[0035] ACGCGCTGGTTATCGATAAAGTTCACCGTAACGATACCGCGGTTTTCAGCTGCTTCGCGTGGAACGAACTGG

[0036] GCAACATCAGCACCACCTTCACCCTGACCGTTATCACCGAAGAAGAAGAAGAACCGGTTGAACAGTCTTAC

[0037] GCGATCTCTAAATGCAACAAAAAAGAAGAAGGCGCGCCGATCTGGAAAACCAGCAAAGAAAACTCTCGTT

[0038] GGATCGCGCGTCCGGCGCGTGCGCCGGTTGAACTGAAATGCCAGGCGTGCGGTAACCCGATCCCGAACATC

[0039] ACCTGGTACAAAAACTCTCAGATCATCGATTCTAGCGTTAAAGAAGGTAAATACCAGGTTGATGGTTACAAA

[0040] CTGAAAATGACCGATCTGACCACCGAAGATAACGGTAACTACACCTGCATGATCCAGAACCGTTACGGTAAA

[0041] CTGATGTGGATGTACATGCTGGAAGTTATCGATCGTAACGTTCAGAAACCGGATGTTGAAGGCCCGGTTAAC

[0042] ACCACCGCGTACATCGGTGAAAACGTTAAACTGCACTGCAAAGTTAAATCTGATATCCAGGCGCACATCCAG

[0043] TGGGTTAAACACTACCAGATCAACGGTACCTACCGTAAACCGGATGGTACCCCGTACGTTTCACGTTATCCAGC

[0044] AGAGCACCTACAACATGACCAACCCGGAAATCCTGGAAATCATCAACGTTAGCCGTAGCGATACCGGTTGGT

[0045] ACACCTGCCTGGTTAGCAACTTCGCGGGCCGTCGTTTCGGTAGCGCGTGGCTGACCGTTGAAGATGTTCCG

[0046] GGTAACGAAAGCCTGAGCAACATGCGTCACGCGCAGGAAGCGCCGCGTAGCTCTGCGAAA (SEQ ID NO: 2).

[0047] Example 2: Preparation of scallop FGFR recombinant protein

[0048] 1) In order to prepare the extracellular region fragment protein of FGFR of SEQ ID NO: 1, a 30-aa tag peptide segment was connected to the N-terminus. The amino acid sequence of the recombinant protein to be expressed with the tag segment connected thereto is as follows:

[0049] HHHHHHSSGLVPRGSHMASMTGGQQMGRGSNDLKVYEGDKARLRCVVKGHPRPWIAWTKNNQSLT

[0050] PKQDPRYKINRNALVIDKVHRNDTAVFSCFAWNELGNISTTFTLTVITEEEEEPVEQSYAISKCNKKEEGAPIWKTS

[0051] KENSRWIARPARAPVELKCQACGNPIPNITWYKNSQIIDSSVKEGKYQVDGYKLKMTDLTTEDNGNYTCMIQN

[0052] RYGKLMWMYMLEVIDRNVQKPDVEGPVNTTAYIGENVKLHCKVKSDIQAHIQWVKHYQINGTYRKPDGTPY

[0053] VHVIQQSTYNMTNPEILEIINVSRSDTGWYTCLVSNFAGRRFGSAWLTVEDVPGNESLSNMRHAQEAPRSSAK

[0054] (SEQ ID NO:3),

[0055] The nucleotide sequence of its corresponding coding gene is as follows:

[0056] CATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGG

[0057] ACAGCAAATGGGTCGCGGATCCAATGATCTGAAAGTTTACGAAGGTGATAAAGCGCGTCTGCGTTGCGTTG

[0058] TTAAAGGCCACCCGCGTCCGTGGATTGCGTGGACCAAAAACAACCAGAGCCTGACCCCGAAACAAGATCC

[0059] GCGTTACAAAATCAACCGTAACGCGCTGGTTATCGATAAAGTTCACCGTAACGATACCGCGGTTTTCAGCTG

[0060] CTTCGCGTGGAACGAACTGGGCAACATCAGCACCACCTTCACCCTGACCGTTATCACCGAAGAAGAAGAAG

[0061] AACCGGTTGAACAGTCTTACGCGATCTCTAAATGCAACAAAAAAGAAGAAGGCGCGCCGATCTGGAAAACC

[0062] AGCAAAGAAAACTCTCGTTGGATCGCGCGTCCGGCGCGTGCGCCGGTTGAACTGAAATGCCAGGCGTGCG<000013...

[0063] GTAACCCGATCCCGAACATCACCTGGTACAAAAACTCTCAGATCATCGATTCTAGCGTTAAAGAAGGTAAATA

[0064] CCAGGTTGATGGTTACAAACTGAAAATGACCGATCTGACCACCGAAGATAACGGTAACTACACCTGCATGAT

[0065] CCAGAACCGTTACGGTAAACTGATGTGGATGTACATGCTGGAAGTTATCGATCGTAACGTTCAGAAACCGGA

[0066] TGTTGAAGGCCCGGTTAACACCACCGCGTACATCGGTGAAAACGTTAAACTGCACTGCAAAGTTAAATCTGA

[0067] TATCCAGGCGCACATCCAGTGGGTTAAACACTACCAGATCAACGGTACCTACCGTAAACCGGATGGTACCCC

[0068] GTACGTTCACGTTATCCAGCAGAGCACCTACAACATGACCAACCCGGAAATCCTGGAAATCATCAACGTTAG

[0069] CCGTAGCGATACCGGTTGGTACACCTGCCTGGTTAGCAACTTCGCGGGCCGTCGTTTCGGTAGCGCGTGGC

[0070] TGACCGTTGAAGATGTTCCGGGTAACGAAAGCCTGAGCAACATGCGTCACGCGCAGGAAGCGCCGCGTAG

[0071] CTCTGCGAAA (SEQ ID NO: 4).

[0072] The nucleic acid fragment with the sequence of SEQ ID NO: 4 synthesized by Sangon Biotech (Shanghai) Co., Ltd. was ligated to the pET-28a(+) vector to obtain a recombinant expression vector.

[0073] 2) The recombinant expression vector in step 1) was transformed into ArcticExpress (DE3) pRARE2 Escherichia coli (Angyu, Shanghai) and cultured overnight at 37°C in kanamycin-resistant LB solid medium. A single colony containing the pET-28a(+)-Fgfr recombinant plasmid was picked and cultured in 10 mL of kanamycin-resistant LB liquid medium at 37°C and 150 rpm overnight. The colony was then transferred to 200 mL of kanamycin-resistant LB liquid medium at a mass concentration of 1% and cultured at 37°C and 150 rpm until the OD600 value was between 0.6 and 0.8. IPTG was added to a final concentration of 0.2 mmol / L (50 mL of the bacterial solution was removed as a control before induction), and the cells were cultured at 4°C and 150 rpm for 16 h. The cells were collected by centrifugation at 7000g for 15 min, and the supernatant was discarded. The cells were lysed with 4 mL of non-denaturing lysis buffer (1 M NaCl, 25 mM Tris-HCl, 5% glycerol, 1% Triton X-ray diffraction). The supernatant and precipitate were collected by centrifugation at 12000 g for 15 min, and the precipitate was resuspended in 4 mL of non-denaturing lysis buffer. 40 μL of sample and 10 μL of 5× protein loading buffer were mixed, and the mixture was incubated at 99°C for 10 min. SDS-PAGE was performed to verify the correct expression of the protein.

[0074] 3) Purifying the protein using nickel-containing affinity resin Ni-NTA Resin; after adding non-denaturing lysis buffer to equilibrate the purification column, the expression product in step 2) was washed with 0 mM, 5 mM, 10 mM, 20 mM, 50 mM, 80 mM, 100 mM, 200 mM, and 300 mM imidazole in sequence to remove impurities, and then eluted with 500 mM imidazole to obtain the target protein.

[0075] In order to improve the expression efficiency, in step 2), the induction conditions of IPTG final concentration of 0.2 mmol / L and induction temperature of 4°C were changed to IPTG final concentration of 0.5 mmol / L and induction temperature of 4°C; IPTG final concentration of 0.2 mmol / L and induction temperature of 8°C; IPTG final concentration of 0.5 mmol / L and induction temperature of 8°C; IPTG final concentration of 0.2 mmol / L and induction temperature of 12°C; and IPTG final concentration of 0.5 mmol / L and induction temperature of 12°C.

[0076] The recombinant protein of the scallop FGFR protein extracellular domain prepared by recombinant expression was subjected to SDS-PAGE gel electrophoresis analysis, with the voltage adjusted to 80V for 40min and then 120V for 70min. Figure 2 and Figure 3 .

[0077] The concentration of the prepared scallop FGFR protein extracellular domain recombinant protein was determined by BCA method and was 0.55 mg / mL. Figure 4 .

[0078] Example 3: Western Blot Verification of Scallop FGFR Extracellular Domain Recombinant Protein Antibody

[0079] The recombinant protein obtained in Example 2 was used as an antigen for animal immunization to prepare the corresponding antibodies. The rabbit polyclonal antibody was synthesized by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. The recombinant protein of the extracellular region of the scallop FGFR protein in Example 2 was subjected to SDS-PAGE gel electrophoresis at a voltage of 80V for 40 minutes and then 120V for 70 minutes; a transfer system was used, adjusting 300mA for 60 minutes to transfer to a PVDF membrane; non-specific sites were blocked with TBST containing 5% skim milk powder; the primary antibody (Anti-FGFR rabbit polyclonal antibody: TBST containing 5% skim milk powder 1:5000) was diluted with a blocking solution containing 5% skim milk powder and incubated at room temperature for 2 hours; the secondary antibody (GoatAnti-Rabbit IgG Antibody: TBST containing 5% BSA 1:5000) was diluted and incubated at 4°C overnight; an ECL chemiluminescence kit was used to perform chemiluminescence detection in an imager. The results are as follows. Figure 5 shown.

[0080] Example 4: Immunohistochemical Validation of Scallop FGFR Extracellular Domain Recombinant Protein Antibody

[0081] Paraffin sections of scallop testes were deparaffinized and rehydrated, then incubated in methanol containing 3% hydrogen peroxide for 30 minutes to quench endogenous peroxidase activity. Antigen retrieval was performed by incubating sections in 1 mmol / L EDTA at 85°C for 40 minutes. To prevent nonspecific binding, sections were incubated in 10% goat serum and 1% BSA in PBS. A rabbit polyclonal antibody (1:300) against the recombinant protein of the scallop FGFR extracellular domain was added to the sections and incubated overnight at 4°C. Sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Sangon Biotech, 1:1000) for 1 hour and then stained with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin. After dehydration with graded alcohol, sections were mounted and observed under a Nikon ECLIPSE Ni microscope.

[0082] See also Figure 6 The rabbit polyclonal antibody against the recombinant protein prepared by the present invention can specifically detect scallop spermatogonia, proving that the recombinant protein can be used as a specific marker for scallop spermatogonia.

[0083] Example 5: Immunofluorescence verification of scallop FGFR extracellular domain recombinant protein antibody

[0084] Cut the testicles of growing male scallops into 1mm pieces 3 The cells were evenly smeared at a concentration of 10 5 The smears were thoroughly dried at 37°C and fixed with 4% paraformaldehyde for 10 minutes at room temperature. The smears were incubated with 1% BSA in PBS at room temperature for 30 minutes, and then incubated with a diluted rabbit polyclonal antibody against the recombinant protein of the FGFR extracellular domain (1:300) in 1% BSA in PBS at room temperature for 1 hour. The smears were washed three times with PBS, each for 5 minutes. ABflo 594-conjugated Goat Anti-Mouse IgG (H+L) (ABclonal, Wuhan) was then diluted 1:200 and incubated at room temperature in the dark for 1 hour. The smears were washed three times with PBS, each for 5 minutes. The slides were mounted with anti-fluorescence quenching mounting solution containing DAPI (Biyuntian, Shanghai). Fluorescence images were acquired using a Zeiss LSM980 Airyscan2 confocal microscope.

[0085] See also Figure 7 The recombinant protein rabbit polyclonal antibody prepared by the present invention can specifically bind to the FGFR protein on the surface of scallop spermatogonia, proving that the recombinant protein can be used as a specific surface marker of scallop spermatogonia.

[0086] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any changes or substitutions within the technical scope of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of a scallop spermatogonia surface marker protein as a specific marker in marking scallop spermatogonia; the amino acid sequence of the scallop spermatogonia surface marker protein is SEQ ID NO:

1.

2. A method for detecting scallop spermatogonia, characterized in that: The method uses immunohistochemistry to detect whether there is a scallop spermatogonia surface marker protein with an amino acid sequence of SEQ ID NO: 1.

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